Intermingled
R Ronzano
20/09/2022
knit: (function(input_file, encoding) { out_dir <- ‘File’; rmarkdown::render(input_file, encoding=encoding, output_file=file.path(dirname(input_file), out_dir, ‘index.html’))})
Figure 2
Figure 2: Double injections of flexor and extensor muscles shows no segregation of premotor interneurons. C: Projections along the transverse (left), sagittal (middle) and coronal (right) axes throughout the lumbar region of the sample showed in A and B (170427n2, UCL). Dots denote individual premotor interneurons, triangles denote infected motor neurons. Convolved density along each axe are shown to the sides of the raw data (top-bottom and left-right distributions in all panels sum to 1). For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments.
Figure 3
Figure 3: Consistent distribution of flexor and extensor premotor interneurons across all individual experiments. A, B: Distribution of premotor interneurons of LG (A) and TA (B) for all the injections. Distributions for each individual experiment are represented with different shades of blue and orange. C: All experiments (single or double ΔG-RabV injections) pooled, showing an overlap of the flexor and extensor related distribution in all quadrants of the spinal cord, with individual dots replaced by contours. For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. D: Correlation (top right) and absolute value of Hedges’ G coefficient (bottom left) across all pairs of experiments, indicating a high degree of consistency and small effect sizes across all experiments, independent of the muscle injected.
Left: Box and whisker plot of the mediolateral position of dorsal ipsilateral premotor interneurons in each experiment. Right: Values of the median for each LG and TA experiment. Pooled distributions split into lumbar segments are shown in Figure 3, figure supplement 1.
Figure 3 figure supplement 1
Figure 3 figure supplement 1: The distribution of premotor interneurons is similar throughout the lumbar spinal cord: data pooled from 18 experiments (11 LG and 7 TA injections) show that within each lumbar segment, from L1 to L6, the distributions of LG and TA premotor interneurons are overlapping.
Figure 3 figure supplement 2
Figure 3 figure supplement 2: Same data as in Figure 3-figure supplement 1 shown before normalization procedures, with idealized spinal cord section scaled to the average size of each segment. Using the raw coordinates, the distribution of LG and TA premotor interneurons are consistently overlapping throughout the lumbar segments.
Figure 3 figure supplement 3
Figure 3 figure supplement 3: distribution of flexor and extensor premotor interneurons pooled across all LG and TA injections shown in the transverse plane (left) and as front (middle) and lateral (right) view along the rostrocaudal axis.
Figure 4
Figure 4: Injection with diluted RabV do not reveal any segregation between flexor and extensor premotor interneurons. A and B show the distribution of LG and TA premotor interneurons on the transverse plane for individual experiments, represented with different colour shades. For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. C: LG and TA distributions overlap and the premotor interneuron distributions are highly correlated across experiments and absolute value of Hedges’ G coefficients are small (D).
Left: Box and whisker plot of the mediolateral position of dorsal premotor interneurons in each experiment. Right: Values of the median for each LG and TA experiment.
Figure 4 figure supplement 1
Figure 4 figure supplement 1: distribution of flexor and extensor premotor interneurons pooled across all LG and TA injections with diluted RabV shown in the transverse plane (left) and as front (middle) and lateral (right) view along the rostrocaudal axis.
Figure 4 figure supplement 2
Figure 4 figure supplement 2: High and low efficiency infections give rise to the same premotor interneurons distributions. Comparison of high and low titre injections are shown in A and E for LG and TA respectively. For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. Correlations between high and low titre experiments are high and effect sizes are low for both LG and TA muscles (B and F) The distributions are similar across experiments for both muscles (B and E) and the median values of the distributions in the ipsilateral dorsal quadrant are not different for high and low efficiency of infection (C and F).
Figure 4 figure supplement 3
data <- data_all %>% filter(lab != "Salk")
animals <- distinct(data.frame(data$animal))
Animal <- as.character(animals$data.animal)
data_fig4supp3 <- data.frame(animal = c(), titer = c(), nb_MN = c(), nb_IN = c(), muscle = c())
for (i in 1:length(Animal)){
df2 <- data %>% filter(animal == Animal[i], identity == "MN")
df3 <- data %>% filter(animal == Animal[i], identity == "IN")
muscles <- distinct(data.frame(df3$muscle))
Muscle <- as.character(muscles$df3.muscle)
for (j in 1:length(Muscle)){
df3m <- df3 %>% filter(muscle == Muscle[j])
df2m <- df2 %>% filter(muscle == Muscle[j])
df3m <- data.frame(lapply(df3m, as.character), stringsAsFactors=FALSE)
if (df3m$lab[1] == "UCL"){
MNs <- nrow(df2m)*2
INs <- nrow(df3m)*2
titer_exp <- df3m$titer[1]
}
if (df3m$lab[1] == "UoG"){
MNs <- nrow(df2m)*8
INs <- nrow(df3m)*8
titer_exp <- df3m$titer[1]
}
if (df3m$lab[1] == "MDC"){
MNs <- nrow(df2m)
INs <- nrow(df3m)
}
v <- c(Animal[i], titer_exp, as.numeric(MNs), as.numeric(INs), Muscle[j])
data_fig4supp3 <- rbind(data_fig4supp3, v, stringsAsFactors = FALSE)
}
}
colnames(data_fig4supp3) <- c("animal", "titer", "nb_MN","nb_IN", "muscle")
data_fig4supp3$nb_MN <- as.numeric(as.character(data_fig4supp3$nb_MN))
data_fig4supp3$nb_IN <- as.numeric(as.character(data_fig4supp3$nb_IN))
data_fig4supp3 <- data_fig4supp3 %>% filter(nb_MN != 0)
#model_log_transformed <- lm(formula = log(nb_MN) ~ log(nb_IN), data = data_fig4supp3)
#fit_log <- summary(model_log_transformed)
#model_linear <- lm(formula = nb_MN ~ nb_IN, data = data_fig4supp3)
#fit_log
graph_fig4supp3 <- ggplot(data_fig4supp3, aes(x = nb_MN, y = nb_IN)) +
#stat_smooth(aes(linetype = "linear"), method = "glm",
#formula = y ~ x, se = TRUE, color = "#145DA0", size = 1, fill = "#145DA0", alpha = 0.2) + #If you want to plot a linear model on non-log transformed data on the same graph
geom_point(aes(shape = titer, color = titer), size = 2) +
geom_smooth(aes(fill = "#76B947"), linetype = "solid", method.args = list(family = gaussian(link = 'log')), method = "glm",
formula = y ~ log(x), se = TRUE, color = "#76B947", size = 1, alpha = 0.2) +
scale_linetype(name = NULL, guide = "legend") +
scale_fill_identity(name = "fitted models", guide = "legend", labels = c("log-transformed")) +
scale_color_manual(values = c("#979797", "#3B3B3B")) +
coord_trans(x="log10", y="log10") +
guides(fill = guide_legend(order = 1)) +
scale_y_continuous(breaks = trans_breaks("log10", function(x) 10^x),
labels = trans_format("log10", math_format(10^.x)), expand = c(0.05,0.05)) +
scale_x_continuous(expand = c(0.05,0.05), breaks = trans_breaks("log10", function(x) 10^x),
labels = trans_format("log10", math_format(10^.x))) +
#stat_regline_equation(label.y = 10^4, aes(label = ..eq.label..)) + #If you want the equation of the model on the figure
#stat_regline_equation(label.y = 10^4-2000, aes(label = ..rr.label..)) + #if you want the R^2 on the fig
ylab("Input INs") +
xlab("Starter MNs") +
theme(axis.text=element_text(size=12, colour = "black")) +
theme_classic() +
theme(axis.line = element_line(colour = "black", size = 0.5, linetype = "solid")) +
theme(axis.text = element_text(colour = 1, size = 12),
legend.background = element_blank(), legend.spacing.y = unit(0.15, 'cm'),
legend.margin = margin(2, 2, 2, 2),
legend.box.background = element_rect(colour = "black"), legend.position = c(0.87, 0.25))
graph_fig4supp3
Figure 4 figure supplement 3: the relation between the number of primary infected motor neurons and premotor interneurons follow a power law. Motor neuron and interneuron numbers in each experiment are shown for high (red) and low (black) titre experiments. The fitted line represents a power law.
Figure 5
Figure 5: Pairs of flexor or extensor muscles show similar distributions of premotor interneurons. A, comparison of pooled data from extensor muscles LG and MG injections. B: correlation and absolute value of Hedges’ G coefficients across all experiments.
Left: box and whisker plots of the mediolateral position of dorsal ipsilateral premotor interneurons for each experiment and distribution of median values (right).
E: similar plot as A, showing the distribution of premotor interneurons following injections of the flexor muscles TA and PL. Correlations and absolute value of Hedges’ G coefficients across each experiment are shown in F.
Mediolateral distribution and positions of the median for each experiment, respectively. For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments.
Figure 6
Figure 6: GlyT2on and GlyT2off premotor interneurons are distributed similarly for TA and LG. A, B: Distribution of GlyT2off (A) and GlyT2on (D) premotor interneurons following LG and TA injections pooled from 4 LG and 3 TA experiments in GlyT2-EGFP::ChAT-Cre mice crossed with RFGT mice, indicating that neither class of premotor interneurons is segregated across muscles. Boxplots and violin plots (B for GlyT2off and E for GlyT2on) show uniformity of distribution across experiments in both the dorsal (top) and ventral (bottom) halves of the cord. C (GlyT2off) and F (GlyT2on) show boxplots and individual values for the medians of the mediolateral distributions restricted to dorsal (top) or ventral (bottom) part of the cord. Ventral premotor GlyT2off and GlyT2on interneurons are differentially distributed. Comparison of excitatory and inhibitory premotor interneurons in LG (G) and TA (J) muscles are similar in the dorsal cord, but differ in the ventral cord, where most ipsilateral premotor interneurons are GlyT2on, and the majority of contralateral premotor interneurons are GlyT2off. Boxplots and violin plots of individual experiments are shown in H for LG and K for TA, highlighting the mediolateral differences in the ventral cord. The medians of the ventral and dorsal distributions are shown in I for LG and L for TA. For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments.
Figure 7
Figure 7: The distribution of flexor and extensor premotor INs is similar in Olig2::Cre; RGT. C - E: Overlay of individual GS (C) and TA (D) experiments and pooled experiments (E). For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. F: Correlation coefficients and absolute value of hierarchical bootstrapped Hedges’ G effect sizes between injections of different muscles and using a different driver for Cre expression.
Box and whisker plots of median values of all the medio-lateral distributions.
Figure 7 figure supplement 1
Figure 7 figure supplement 1: distribution of flexor and extensor premotor interneurons pooled across GS and TA injections performed in Olig2::Cre; RFGT mice. Distributions are shown in the transverse plane (left) and as front (middle) and lateral (right) view along the rostrocaudal axis.
Figure 8
Figure 8: Injection of AAV-G and RabV into GS and TA muscles does not reveal segregation of premotor interneurons. (C). For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. Individual experiments are highly correlated and have low absolute value of Hedges’ G effect sizes (D).
Box and whisker plots of median values of all the medio-lateral distributions.
Figure 9
Figure 9: Injection of AAV-FLEX-G in ChAT::Cre mice gives rise to overlapping distributions of flexor and extensor related premotor interneurons. The pooled distributions of flexor and extensor related infected neurons are similar on the transverse and longitudinal planes (C). For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. There is strong correlation and low effect sizes across individual experiments (D).
Box and whisker plots of median values of all the medio-lateral distributions with identical values for flexor and extensor injections.
Figure 10
Figure 10: Injection of AAV-FLEX-G prevents anterograde spreading of RabV from afferent terminals. A: side by side comparison of pooled transverse distribution of infected interneurons from GS (left) and TA (right) with AAV-G or AAV-FLEX-G and RabV. The distributions obtained with AAV-G show a marked mediolateral shift and extensive labelling of superficial dorsal horn neurons, a feature that is absent when anterograde transfer is prevented by restricting the expression of the G protein to motor neurons only. The distributions obtained are very similar across the different muscles, but the mediolateral shift is reflected in the lower correlation values and high hierarchical bootstrapped effect size (B) between AAV-G and AAV-FLEX-G injections.
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Medians evidence the mediolateral shift obtained the different methods, AAV-G and AAV-FLEX-G injections.
Figure 11
Figure 11: Injections of PRV-Bartha in GS and TA muscles give rise to similar distribution of premotor interneurons. The distributioof premotor interneurons are similar along all axis (C). For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. Individual experiments are highly correlated, with small effect size (D).
Box and whisker plots of median values of all the medio-lateral distributions with identical values for flexor and extensor injections.
Figure 12
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Figure 12: Regardless of the tracing methods, flexor and extensor related premotor interneurons distributions are always overlapping. The correlation and absolute value of hierarchical bootstrapped Hedges’ G coefficient matrix across pooled experiments obtained with different injection strategies exhibits high values of correlation  and low effect sizes across different techniques and across the two different muscles (A). The median values of the mediolateral position of premotor interneurons are similar for all conditions (B).
Figure 12 figure supplement 1
Figure 12 figure supplement 1: Distribution of motor neurons (triangles) and premotor interneurons (dots) of 3 LG (blue) and TA (yellow) double injections (UCL) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 2
Figure 12 figure supplement 2 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 2 LG (blue) and TA (yellow) double injections (UoG) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 3
Figure 12 figure supplement 3 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 2 LG (blue) and MG (dark blue) double injections (UCL) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 4
Figure 12 figure supplement 4 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 4 LG (blue) and MG (yellow) double injections (UoG) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 5
Figure 12 figure supplement 5 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 2 TA (yellow) and PL (dark orange) double injections (UCL) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 6
Figure 12 figure supplement 6 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 4 MG (dark blue) single injections (UoG) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 7
Figure 12 figure supplement 7 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 3 PL (dark orange) single injections (UoG) of rabies virus in ChAT::Cre+/-; RFGT+/- mice.
Figure 12 figure supplement 8
Figure 12 figure supplement 8 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 3 LG (blue) and TA (yellow) double injections with low titre rabies virus in ChAT::Cre+/-; RFGT+/- mice (UoG).
Figure 12 figure supplement 9
Figure 12 figure supplement 9 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 4 LG (blue) single injections with low titre rabies virus in ChAT::Cre+/-; RFGT+/- mice (UoG).
Figure 12 figure supplement 10
Figure 12 figure supplement 10 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 3 TA (yellow) single injections with low titre rabies virus in ChAT::Cre+/-; RFGT+/- mice (UoG).
Figure 12 figure supplement 11
Figure 12 figure supplement 11 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 4 GS (blue) single injections with rabies virus in Olig2::Cre +/-; RFGT+/- mice (MDC).
Figure 12 figure supplement 12
Figure 12 figure supplement 12 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 3 TA (yellow) single injections with rabies virus in Olig2::Cre+/-; RFGT+/- mice (MDC).
Figure 12 figure supplement 13
Figure 12 figure supplement 13 Distribution of infected neurons (primary infected motor neurons or secondary infected interneurons are not distinguished) of 2 GS (blue) and TA (yellow) double injections and 2 GS single injections of rabies and AAV-Ef1a-B19G in wild type mice (Salk).
Figure 12 figure supplement 14
Figure 12 figure supplement 14 Distribution of infected neurons (primary infected motor neurons or secondary infected interneurons are not distinguished) of 4 GS (blue) single injections of rabies and AAV-CAG-Flex-oG in Chat::Cre+/- mice (Salk).
Figure 12 figure supplement 15
Figure 12 figure supplement 15 Distribution of infected neurons (primary infected motor neurons or secondary infected interneurons are not distinguished) of 3 TA (yellow) single injections of rabies and AAV-CAG-Flex-oG in Chat::Cre+/- mice (Salk).
Figure 12 figure supplement 16
Figure 12 figure supplement 16 Distribution of infected neurons (primary infected motor neurons or secondary infected interneurons are not distinguished) of 4 GS (blue) and TA (yellow) double injections of PRV-152 and PRV-614 in wild type mice (Salk).
Figure 13
